1. Field of the Invention:
The present invention relates to in vivo expression technology, and more particularly to a method of identifying and studying genes that display temporal or spatial patterns of expression during infection of host-tissues.
2. Description of the State of the Art:
An infection of the human body by a pathogen, or disease-producing microorganism, results in disease when the potential of the microorganism to disrupt normal bodily functions is fully expressed. Some disease-producing microorganisms possess properties, referred to as virulence factors, that enhance their pathogenicity and allow them to invade host or human tissues and disrupt normal bodily functions. The virulence of pathogens, that is, their ability to induce human disease, depends in large part on two properties of the pathogen, invasiveness and toxigenicity. Invasiveness refers to the ability of the pathogen to invade host or human tissues, attach to cells, and multiply within the cell or tissues of the human body. Toxigenicity refers to the ability of a pathogen to produce biochemicals, known as toxins, that disrupt the normal functions of cells or are generally destructive to cells and tissues.
Scientists can develop better therapeutic and diagnostic approaches against pathogenic microbes if they understand better the molecular mechanisms of the specific pathogenic microbes or microorganisms that allow them to circumvent the host's, e.g., human body, immune system and initiate the physiological changes inherent in the disease process. To do so, scientists must identify those virulence factors, or microbial gene products, that are specifically required for each stage of the infection process. Environmental conditions within the host are responsible for regulating the expression of most known virulence factors (J. Mekalanos, 1992, J. Bacteriol. 174:1). Consequently, scientists attempt to mimic, in vitro, the environmental conditions within the host in an attempt to identify and study those genes that encode and are responsible for producing virulence factors. As a result, the ability to identify and study the expression of many virulence factors has been dependent on, and limited by, the ability of researchers to mimic host environmental factors in the laboratory.
There have been some methods developed for identifying and studying the expression of virulence genes of microorganisms involved in pathogenesis. For example, a method referred to as insertional mutagenesis has long been recognized as a technique to inactivate and identify genes. Insertional mutagenesis relies on the ability of short stretches of DNA, known as insertion sequences, to move from one location to another on a chromosome by way of nonreciprocal recombination. Insertion sequences are not homologous with the regions of the plasmid or the chromosome into which it is inserted. Therefore, independent mutational events may be generated by randomly inserting an insertion sequence into a gene, thereby, disrupting the expression of that gene. As each mutated gene represents a different case, the selection procedure utilized in successfully recovering insertional mutants is critical. In vitro assays are designed to screen for insertional activation events, i.e., the turning "on" of a previously silent gene, or insertional inactivation events, i.e., the turning "off" of a previously expressed gene. For an example of the insertional mutagenesis method. Fields, et al., 1986, Proc. Natl. Acad. Sci. USA 83:5189-5193.
The second basic technique utilized in the identification of genes is referred to as a cloning screen. Essentially, a piece of DNA or gene from the organism of interest is spliced into either a plasmid or a lambda phage, referred to as the vehicle or vector, and the resulting chimefie molecule is used to transform or infect, respectively, a host cell. A determination is then made as to whether the piece of DNA or gene of interest is capable of conferring a specific phenotype to the host cell which it would not otherwise possess, but for the gene of interest. For example, in a technical paper (R. Isberg, et al., "A Single Genetic Locus Encoded by Yersinia pseudotuberculosis Permits Invasion of Cultured Animal Cells by Escherichia coli K-12," Nature, 1985, 317:262-264) a cloning screen is disclosed in which a cosmid clone bank is prepared from Y. pseudotuberculosis genomic DNA and introduced into a bacterial E. coli K-12 strain. The E. coli K-12 strain containing random sequences of DNA representing the entire genetic information for Y. pseudotuberculosis was pooled, grown in broth, i.e., a complete medium, and used to infect a monolayer of cultured HEp-2 cells, i.e., animal cells. The cultured animal cells were then cultured and tested to determine whether introducing DNA from Y. pseudotuberculosis to E. coli confers an invasive phenotype typical of Y. pseudotuberculosis to E. coli.
A third method discussed by A. Osbourn, et al., entitled "Identification of plant induced genes of the bacterial pathogen Xanthornonas campestris pathovar campestris using a promoterprobe plasmid" (EMBO J., 1987, 6:23-28) discloses a promoter probe plasmid for use in identifying promoters that are induced in vivo. Random chromosomal DNA fragments are cloned into a site in front of a promoterless chloramphenicol acetyltransferase gene contained on a plasmid. Transconjugates were then produced by transferring the resulting library into Xanthornonas. Individual transconjugates are then introduced into chloramphenicol-treated seedlings to determine whether the transconjugate displays resistance to chloramphenicol in the plant and then on an agar plate.
The final method utilized in the identification of genes is referred to as a regulatory screen. S. Knapp, et al., in his technical publication, entitled "Two Trans-Acting Regulatory Genes (vir and rood) Control Antigenie Modulation in Bordetella pertussis," (J. Bacteriol, 1988, 170:5059-5066) discloses a method for identifying potential virulence genes based on their coordinate expression with other known virulence genes under defined laboratory conditions.
Transcriptional fusions to heterologous reporter genes such as .beta.-galactosidase and chloramphenicol-acetyl transferase have been extensively used to study gene expression in both prokaryotic and eukaryotic organisms (Slauch, J. M. & Silhavy, T. J., 1991, Methods Enzymol. 204:212-248). Although they provide an elegant method in most instances, these reporter fusion systems are technically limited to assaying expression within large populations of the test organism and are at times dictated by the reporter half-life. These limitations are particularly troublesome when complex systems are being probed, i.e., studying gene expression by pathogenic microbes during infection of a host organism.
The above technical papers by Fields, et al., R. Isberg, et al, and S. Knapp, et al., each disclose methods for identifying microorganismal genes; however, the selection procedures or in vitro assays utilized in each method depends upon the ability of the in vitro assay to mimic the environmental conditions within the host, i.e., the in vivo environmental conditions. A disadvantage of these approaches is that each requires some understanding of the environmental conditions necessary to obtain virulence gene expression. Furthermore, the ability to study the spatial and temporal expression of these genes is also limited. Consequently, scientists have resorted to mixing host cells with the pathogen of interest in vitro to approximate the host's environmental conditions. Short of an exact duplication of the host's environmental conditions, critical regulatory factors necessary for the expression of many virulence factors may be missing, thus making the identification and study of those genes responsible for encoding virulence factors impossible.
While the technical paper by A. Osbourn, et al., discloses a method to screen for promoters that are induced in vivo, a disadvantage is that no feasible method exists to select genes of a particular class, that is, individual transconjugates must be screened one by one in individual seedlings to determine whether a promoter is inducible. A further disadvantage results from a phenomena referred to as a position effect. A. Osbourn, et al., utilizes an autonomous plasmid and therefore the regulation of the promoter may vary considerably from the regulation of the promoter as it is found in its natural environment on the Xanthomonas genome. Other complications that arise from the use of plasmids are copy number, stability and supercoiling effects.
There is still a need, therefore, for a method or technique for identifying and studying genes encoding virulence factors in their normal environment whose expression is regulated, or turned "on", by undetermined factors within the host.